Dataset Information

MicroRNA-205 mediates endothelial progenitor functions in distraction osteogenesis by targeting the transcription regulator NOTCH2.

ABSTRACT:

Background

Distraction osteogenesis (DO) is a highly efficacious form of reconstructive bone regeneration, but its clinical utility is limited by the prolonged period required for bone consolidation to occur. Understanding the mechanistic basis for DO and shortening this consolidation phase thus represent promising approaches to improving the clinical utility of this procedure.

Methods

A mandibular DO (MDO) canine model was established, after which small RNA sequencing was performed to identify relevant molecular targets genes. Putative miRNA target genes were identified through bioinformatics and confirmed through qPCR, Western blotting, and dual-luciferase reporter assays. Peripheral blood samples were collected to isolate serum and endothelial colony-forming cells (ECFCs) in order to measure miR-205, NOTCH2, and angiogenic cytokines expression levels. Lentiviral constructs were then used to inhibit or overexpress miR-205 and NOTCH2 in isolated ECFCs, after which the angiogenic activity of these cells was evaluated in migration, wound healing, proliferation, tube formation, and chick chorioallantoic membrane (CAM) assay. Autologous ECFCs transfected to knockdown miR-205 and were injected directly into the distraction callus. On days 14, 28, 35 and 42 after surgery, bone density was evaluated via CBCT, and callus samples were collected and evaluated via histological staining to analyze bone regeneration and remodeling.

Results

MiR-205 was identified as being one of the miRNAs that was most significantly downregulated in MDO callus samples. Downregulation of miR-205 was also observed in DO-ECFCs and serum of animals undergoing MDO. Inhibiting miR-205 markedly enhanced angiogenesis, whereas overexpressing miR-205 had the opposite effect in vitro. Importantly, NOTCH2, which is a unique regulator in bone angiogenesis, was identified as a miR-205 target gene. Consistent with this regulatory relationship, knocking down NOTCH2 suppressed angiogenesis, and transduction with a miR-205 inhibitor lentivirus was sufficient to rescue angiogenic activity. When ECFCs in which miR-205 had been inhibited were transplanted into the MDO callus, this significantly bolstered osteogenesis, and remodeling in vivo.

Conclusions

MiR-205 is a significant regulator of the MDO process, and inhibiting this miRNA can accelerate MDO-related mineralization. Overall, these results offer new insights into the mechanistic basis for this procedure, highlighting potential targets for therapeutic clinical intervention.

SUBMITTER: Jiang W

PROVIDER: S-EPMC7860583 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

MicroRNA-205 mediates endothelial progenitor functions in distraction osteogenesis by targeting the transcription regulator NOTCH2.

Jiang Weidong W Zhu Peiqi P Zhang Tao T Liao Fengchun F Yu Yangyang Y Liu Yan Y Shen Huijuan H Zhao Zhenchen Z Huang Xuanping X Zhou Nuo N

Stem cell research & therapy 20210203 1

<h4>Background</h4>Distraction osteogenesis (DO) is a highly efficacious form of reconstructive bone regeneration, but its clinical utility is limited by the prolonged period required for bone consolidation to occur. Understanding the mechanistic basis for DO and shortening this consolidation phase thus represent promising approaches to improving the clinical utility of this procedure.<h4>Methods</h4>A mandibular DO (MDO) canine model was established, after which small RNA sequencing was perform ...[more]

PMID: 33536058

Similar Datasets

Project description:BackgroundDistraction osteogenesis (DO) is a widely used bone regenerative technique. However, the DO process is slow, and the consolidation phase is long. Therefore, it is of great clinical significance to explore the mechanism of DO, and shorten its duration. Recent studies reported that stem cell exosomes may play an important role in promoting angiogenesis related to DO, but the mechanism remains unclear.MethodsCanine endothelial colony-forming cells (ECFCs) were isolated and cultured, and the expression of THBS1 in canine ECFCs were inhibited using a lentiviral vector. The exosomes secreted by canine ECFCs were isolated and extracted, and the effect of exosomes on the angiogenic activity of Human umbilical vein endothelial cells (HUVECs) was detected by proliferation, migration, and tube formation experiments. WB and qRT-PCR were used to explore the effects and mechanisms of THBS1-mediated ECFC-Exos on HUVECs angiogenesis. Then, a mandibular distraction osteogenesis (MDO) model was established in adult male beagles, and exosomes were injected into the canine peripheral blood. Micro-CT, H&E, Masson, and IHC staining were used to explore the effects and mechanisms of THBS1-mediated ECFC-Exos on angiogenesis and osteogenesis in the DO area.ResultsECFC-Exo accelerated HUVECs proliferation, migration and tube formation, and this ability was enhanced by inhibiting the expression of THBS1 in ECFC-Exo. Using Western blot-mediated detection, we demonstrated that inhibiting THBS1 expression in ECFCs-Exo activated PI3K, AKT, and ERK phosphorylation levels in HUVECs, which promoted VEGF and bFGF expressions. In the DO model of the canine mandible, ECFCs-Exo injected into the peripheral blood aggregated into the DO gap, thus promoting angiogenesis and bone formation in the DO tissue by reducing THBS1 expression in ECFC-Exo.ConclusionOur findings suggested that ECFC-Exos markedly enhances angiogenesis of endothelial cells, and promotes bone healing in canine MDO. Thus, THBS1 plays a crucial role in the ECFC-Exos-mediated regulation of canine MDO angiogenesis and bone remodeling.The translational potential of this articleThis study reveals that the angiogenic promotion via THBS1 suppression in ECFC-Exos may be a promising strategy for shortening the DO duration.

Project description:Background: Interfragmentary movements have benefits in the improvement of bone formation during distraction osteogenesis (DO). Although several clinical studies reported positive outcomes regarding the application of the cyclic distraction-compression (CDC) dynamization technique in cases with poor bone formation during DO, they are mostly anecdotal without a detailed description. The purpose of this study was to investigate the effectiveness and potential mechanism of different amplitudes and rates of the CDC technique on bone regeneration in a rat femur DO model. Methods: A total of 60 adult male Sprague-Dawley rats underwent right femoral mid-diaphysis transverse osteotomy and were randomly and evenly divided into Control (no manipulation), Group1 (CDC therapy), Group2 (CDC therapy with larger amplitude), and Group3 (CDC therapy with a slower rate) after distraction. The CDC technique was performed during the middle phase of the consolidation period according to different protocols. Animals were sacrificed after 4 and 6 weeks of consolidation. The process of bone formation was monitored by digital radiographs, and the regenerate bone was evaluated by micro-computed tomography (micro-CT), biomechanical test, and histological analysis. The serum contents of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were measured by enzyme-linked immunosorbent assay (ELISA). Results: Bone regeneration after the CDC technique was improved significantly during DO. The digital radiograph, micro-CT, histomorphological analysis, and biomechanical evaluation showed better effects regarding volume, continuity, and mechanical properties of the regenerate bone in Group2 and Group3 when compared to Group1. The angiogenic and osteogenic markers were more highly expressed in Group2 and Group3 than in Group1 according to the immunohistochemical analysis. As for ELISA, the serum contents of HIF-1α and VEGF were also increased after the CDC technique, especially in Group2 and Group3. Conclusion: The CDC dynamization technique has benefits on the improvement of bone formation during DO, and the mechanism may be due to tissue hypoxia activating the HIF pathway followed by the augmentation of osteogenic-angiogenic coupling. Better outcomes may be achieved by moderately increasing the amplitude and slowing down the rate of the CDC technique.

Dataset Information

MicroRNA-205 mediates endothelial progenitor functions in distraction osteogenesis by targeting the transcription regulator NOTCH2.

Background

Methods

Results

Conclusions

Publications

MicroRNA-205 mediates endothelial progenitor functions in distraction osteogenesis by targeting the transcription regulator NOTCH2.

Similar Datasets

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